Biotin-binding containment systems

ABSTRACT

The present invention relates to genetic containment systems which express a biotin-binding component that can be used for selectively destroying recombinant cells such as genetically engineered microorganisms. These systems may comprise a streptavidin or an avidin gene whose expression is controlled by a regulatable promoter. The regulatory agent such as a transcriptional effector is expressed from another gene which may also be expressed and its expression controlled by the containment system. Expression of the agent can be designed to respond to physiological changes in the environment. The invention also relates to containment systems and methods for the selective detection or tracking of recombinant cells and to eukaryotic and prokaryotic cells which contain these genetic containment systems.

RIGHTS IN THE INVENTION

This invention was made with United States Government support, undergrant number DAAH04-94-2-0004, awarded from the United States Departmentof the Army, and the United States government has certain rights in theinvention.

BACKGROUND

1. Field of the Invention

This invention relates to genetic systems for the containment ofrecombinant organisms and to cells which possess these systems. Theinvention also relates to methods for the selective detection andselective destruction of recombinant cells.

2. Description of the Background

Genetic engineering, although still in its infancy, is increasinglybeing utilized in medicine, agriculture and industry. Geneticallyengineered microorganisms (GEMs), microorganisms which have deliberatelyhad their genetic character in some way directly altered, are among themost common tools for the genetic engineer. These microorganisms, whichmay be prokaryotic or eukaryotic or unicellular or multicellular, areused in the generation of, for example, medical products,insect-resistant crops and healthier food products. GEMs are also widelyused in the elimination of waste products such as biomass, sludge andaccidental spills of oil or toxins.

Surprisingly, little data is available on how GEMs survive in theenvironment or how recombinant DNA can spread among indigenous bacterialpopulations. It is relatively unknown whether recombinant microorganismshave the ability to alter, in a temporary or permanent fashion, anatural ecosystem or any environment which they might be found.Considering the undetermined consequences associated with the release ofnew genetic material into the environment, various techniques have beendeveloped in an effort to destroy GEMs on demand.

As microorganisms are generally biodegradable, when killed, they poselittle risk of damage to the environment. Typically, GEMs are killed anddisposed of through physical or chemical means such as incineration,glassification, solvent extraction, chemical treatments, super criticalfluid extraction, ozonolysis, UV light treatments and many others. Thesemethods are generally expensive and time consuming requiring a greatdeal of labor and physical manipulations. Even incineration is comingunder increasing scrutiny due to concerns with gaseous outputs. Ingeneral, the costs for any of these methods runs in the hundreds to thethousands of dollars per ton of contaminated material due to thehazardous nature of the material, handling issues, residue problemsafter treatment, energy input costs and related issues.

Although these techniques are quite effective, each is based on theprinciple that the GEMs can be physically confined. However, absoluteconfinement is often not possible or simply impractical. More recenttechniques have focused on various methods of biological containment.Potential risks associated with deliberate or unintentional release ofGEMs are minimized by the use of debilitated mutant strains ornon-conjugative, non-mobilized plasmids. GEMs that escape physicalconfinement, according to a preprogrammed genetic design, are destroyedor cannot successfully reproduce. For example, GEMs can be engineered torequire an essential nutrient which is otherwise rare or non-existent inthe natural environment. GEMs can also be programmed to die in thepresence of compounds which are abundant outside of the laboratorysetting. Additional approaches include the introduction of conditionalmaintenance functions into GEMs, so that their survival is dependent onthe specific environments (J. L. Ramos et at., Bio/Technology 13:35-37,1995) or growth phase (P. Klemm et at., Appl. Environ. Microbiol.61:481-86, 1995). Thus, by inserting toxic genes transcribed frompromoters responding to environmental or intracellular changes, theviability of GEMs can be controlled. Unfortunately, the overallefficiency of suicide systems typically becomes reduced over time bymutational inactivation of the lethal cassettes although some systemsbenefited by combining two or more different lethal genes.

The most extensively studied toxic genes represent the Escherichia coligef gene family (hok, gef and relF), whose expression disrupts the cellmembrane potential (A. K. Bej et at., Appl. Environ. Microbiol.54:2472-77, 1988). Other successfully tested genes include Serratiamarcescens and Staphylococcus aureus endonucleases (S. Molin, Curr. Op.Biotech. 4:299-305, 1993), Serratia liquefaciens phospholipase A (S.Molin et al., Annu. Rev. Microbiol. 47:139-66, 1993), and Bacillussubtilis sacB gene which confers sucrose sensitivity (G. C. Recorbet etal., Appl. Environ. Microbiol. 59:1361-66, 1993). Preliminaryexperiments with lysis genes from bacteriophages are also promising (S.Molin et at., Annu. Rev. MicrobioI. 47:139-66, 1993).

Prior studies to control the proliferation of genetically engineeredmicroorganisms have primarily considered two basic approaches. The firstapproach was to use no containment system at all for the engineeredmicroorganism. The assumption was that even if the engineered organismssurvive, they would not disrupt normal ecological balances in theenvironment. The other approach was to develop suicide cassettes tocontrol the survival of the genetically engineered microorganism.Variations include the induction of peptides to disrupt membraneintegrity (A. K. Bej et al., Appl. Environ. Microbiol. 54:2472-77,1988), a TOL plasmid suicide system involving induction of the gef geneto promote cell death (A. Contreras et at., Appl. Environ. Microbiol.57:1504-8, 1991; S. Molin et at., Bio/Technology 5:1315-18, 1987) andinduction of a relF gene to promote cell suicide. However, with theseapproaches, the levels of inducers and the formation of resistanceclones due to high mutation rates, genetic instability, remain asproblems.

SUMMARY OF THE INVENTION

The invention overcomes the problems and disadvantages associated withcurrent strategies and designs and provides novel genetic systems,genetic elements and methods for selectively killing or detectingrecombinant organisms.

One embodiment of the invention is directed to genetic containmentsystems comprising a suicide cassette that encodes a biotin-bindingcomponent. The biotin-binding component, which may be streptavidin, avidor a modification of these proteins, can be either the suicide gene or amarker gene.

Another embodiment of the invention is directed to nucleic acids thatencode genetic containment systems comprised of a gene encoding abiotin-binding product whose expression is controlled by atranscriptional effector also encoded within the nucleic acid. Nucleicacids may be suicide cassettes wherein the suicide gene encodes astreptavidin or avidin protein, or a conventional suicide gene.

Another embodiment of the invention is directed to cells which containgenetic containment systems that encode a biotin-binding component.Cells may be prokaryotic such as a bacterial cell or eukaryotic such asa plant, animal or yeast cell.

Another embodiment of the invention is directed to method forselectively killing a recombinant microorganism. The microorganism istransformed with a nucleic acid which contains a suicide cassette thatencodes a biotin-binding protein such as streptavidin. The cell can bekilled by stimulation of streptavidin which binds and effectivelyeliminates biotin from the cell.

Another embodiment of the invention is directed to methods for thespecific detection of recombinant cells. Cells are transformed with anucleic acid containing a genetic containment system which encodes abiotin-binding protein. Expression of the cassette can be coupled withexpression of the biotin-binding protein. Cells can be contacted with abiotinylated solid support and thereby detected.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, in part, will be obviousfrom this description and may be learned from the practice of theinvention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 Physical map of stv cassette.

FIG. 2 IPTG-induced in vitro containment of E. coil DH5α (pRO-slp).Cultures were grown in the presence of ampicillin,

FIG. 3 IPTG-induced in vitro containment of P. putida KT2440 (pVLT-slp).Cultures were grown in the presence of kanamycin.

DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present invention isdirected to biotin-binding genetic containment systems and torecombinant cells which contain these systems. The invention is alsodirected to methods for the detection and selective destruction ofrecombinant cells using biotin-binding expression systems.

With the advent of recombinant technology, GEMs, or geneticallyengineered microorganisms, are being used with ever increasing frequencyin a wide. array of industrial technologies. Although most pose littledirect concern to the environment or humans, there still exists thepossibility of a unpredicted and detrimental consequence resulting fromthe release of new genetic material into the natural environment.Conventional safety procedures include physical confinement andsubsequent sterilization of all materials associated with the GEMs, orthe incorporation of suicide cassettes into the microorganisms.Unfortunately, physical confinement procedures, when possible, areexpensive and time consuming and generally are not applicable to actualconditions. Further, available genetic containment systems aregenetically unstable or do not operate successfully in the complexconditions encountered in the natural environment. These suicide systemsoften cannot be adapted to specific cells or the specific environmentalconditions being utilized.

The present invention overcomes these and other problems by providing agenetic containment system based on the expression of a biotin-bindingcomponent such as streptavidin, avidin or another biotin-binding geneexpression product. Streptavidin and avidin are each fairly toxic inmost cellular systems. Toxicity is caused by their exceptionally highbinding affinity for biotin, an essential element for a very widevariety of microorganisms including bacteria, plant and animal cells,and even yeast cells. Binding effectively depletes this basic vitaminfrom the cell and kills the host organism or microorganism. The systemis, thus, broadly applicable across class, order, genus and speciesboundaries, and can be used in most situations irrespective ofphysiological conditions in the laboratory or the natural environment.As such, biotin-binding genetic containment systems are useful torestrict the proliferation of GEMs as well as other microorganisms andorganisms in general.

Streptavidin, the preferred biotin-binding component, is a tetramericprotein, having four identical subunits, and is secreted by theactinobacterium Streptomyces avidinii. Both streptavidin, and itsfunctional homolog avidin, exhibit extremely tight and highly specificbinding to biotin which is one of the strongest known non-covalentinteractions (K_(d) ˜10⁻¹⁵ M) between proteins and ligands. Althoughavidin and streptavidin have almost the same high affinity for biotin,they are different in many other respects. The two proteins havedifferent molecular weights, electrophoretic mobilities and overallamino acid composition. Avidin is a glycoprotein found in egg whites andthe tissues of birds, reptiles and amphibia. Like streptavidin, avidinhas almost the same high affinity for biotin and exists as a tetramerwith a molecular weight of between about 67,000 to about 68,000 daltons.Avidin also has a high isoelectric point of between about 10 to about10.5 and contains carbohydrates which cause it to bind non-specificallyto biological materials including cell nuclei, nucleic acids andlectins. These non-specific interactions make avidin less suitable thanstreptavidin for many applications.

The full-length streptavidin monomer is 159 amino acids in length, some30 residues longer than avidin. It contains no carbohydrate and has aslightly acidic isoelectric point of about 6.0 which accounts, in part,for the low non-specific binding level. Each subunit of streptavidin isinitially synthesized as a precursor of 18,000 daltons which forms atetramer of about 75,000 daltons. Secretion and post-secretoryprocessing results in mature subunits having an apparent size of 14,000daltons. Processing occurs at both the amino and carboxyl termini toproduce a core protein of about 13,500 daltons, having about 125 to 127amino acids. This core streptavidin forms tetramers and binds to biotinas efficiently as natural streptavidin. The mature streptavidin tetramerbinds one molecule of biotin per subunit and the complex, once formed,is unaffected by most extremes of pH, organic solvents and denaturingconditions. Separation of streptavidin from biotin requires conditions,such as 8M guanidine, pH 1.5, or autoclaving at 121° C. for 10 minutes.Mutations of the streptavidin or core streptavidin protein exist wherebybinding affinity is reduced such that dissociation can be more easilyperformed without damage to the attached biotin-bound molecule.

Biotin, also known as vitamin H orcis-hexahydro-2-oxo-1H-thieno-(3,4)-imidazole-4-pentanoic acid, is abasic vitamin which is essential for most organisms including bacteriaand yeast. Its depletion caused by the production of streptavidin incells is potentially lethal. In mammals, the tissues having the highestamounts of biotin are the liver, kidney and pancreas. Biotin levels alsotend to be raised in minors and tumor cells. In addition to cells,biotin can be isolated from secretions such as milk which has a fairlyhigh biotin content. Biotin has a molecular weight of about 244 daltons,much lower than its binding partners avidin and streptavidin. Biotin isalso an enzyme cofactor of pyruvate carboxylase, trans-carboxylase,acetyl-CoA-carboxylase and beta-methylcrotonyl-CoA carboxylase whichtogether carboxylate a wide variety of substrates.

A biotin-binding genetic containment system has numerous advantages overmore conventional approaches. First, the system is based on a differentsuicide function than those previously utilized, namely biotin-binding.Second, the cassette approach described can be used in a wide range ofhosts including bacteria, fungi, algae, higher plants and animal cells.Third, rates of mutation appear to be significantly lower than most ofthose previously reported for other suicide gene and, therefore, thereis better control in environmental settings. Fourth, the suicidecassette can be easily coupled to catabolic plasmids to demonstratefunction. Consequently, genetic constructs tightly couple thedegradation of hazardous chemicals to cell survival. Further, theengineered organisms can be easily detected and monitored withbiotin-bound conjugates due to the production of streptavidin.

One embodiment of the invention is directed to a genetic containmentsystem containing at least one nucleic acid cassette that encodes abiotin-binding component. The component may be used as the suicidefunction or as a marker for subsequent detection of recombinantorganisms. The biotin-binding component is preferably a protein whichspecifically binds to biotin such as streptavidin or avidin, derivativesor mutations of streptavidin or avidin, or combinations of thesecomponents. Biotin-binding components may also be nucleic acids such asRNA, DNA or even PNA sequences that have an affinity for biotin and canbe expressed or otherwise obtained from nucleic acid.

Genetic containment systems of the present invention contain one or moresuicide genes for the pre-programmed death of the recombinant cell.Suicide genes can be conventional genes such as members of the E. coligef gene including hok, gef and relF, the Serratia marcescens andStaphylococcus aureus endonuclease genes, the Serratia liquefaciensphospholipase A gene, the Bacillus subtilis sacB gene, lysis genes frombacteriophages, streptavidin genes, avidin genes, or mutations orcombinations of these genes. The biotin-binding aspect may therefore bethe principle killing function of the system or an ancillary orsecondary feature to supplement another killing function. Consequently,another aspect of the invention is directed to combinations of thebiotin-binding containment system with other types of suicide genecassettes to further extend the killing efficiency of a containmentsystem by reducing mutation rates.

The need for new killing genes involved in regulatory circuits ofprogrammed cell death comes from imperfect killing by one toxinoperating alone. Combinations of at least two different toxic peptidescan drop appearance of mutants to 10⁻¹⁰ and below. For example, thestv-based system shown in FIG. 1, built in the pR01614 vector (pMB1 andpRO1600 replicon), can deliver a second suicide function in constructsalready integrated within chromosome or based on R300B plasmid replicon.This could be performed in P. putida. Streptavidin fulfills allrequirements of a toxin to be involved in killing systems. The rate ofmutational inactivation of stv cassette is at least equal to thoseestimated for constructs based on a single copy gene per plasmid.Accumulation of mutants, for example with P. putida, occurs throughinsufficient repression of the uninduced system because of extremely lowproduction of LacI protein.

In genetic containment systems, expression of the suicide gene should betightly controlled. Unwanted generation of the suicide product can havepremature deadly consequences. In such situations, the system becomesdifficult to replicate and handle in microorganisms and eventuallyuseless for genetic containment. In addition, viable mutations not onlydevelop, but are naturally selected for in every round of reproduction.Expression of the biotin-binding protein can be controlled byfunctionally linking the gene to a regulatable transcriptional promoter,many of which are well-known to those of ordinary skill in the art. Theregulated promoter is preferably a heterologous promoter wherein one ormore of the key regulatory agents of the promoter are absent from thehost cell. In this fashion, those agents can be introduced artificiallyand specifically targeted to stimulate or shut down the promoter.Promoters which are useful include the bacteriophage promoters, SP6, T7,T3 and the γ p_(L) promoters, the amino acid promoters trp, lac, hybridtrp-lac, phoA and gal, and the eukaryotic promoters for metallothioneinpromoters, MMTV promoters, inducible promoters and hybrids andcombinations of these promoters. In addition, variations of thesepromoter sequences can be used in prokaryotic or eukaryotic geneticelements to regulate expression.

Streptavidin-based containment systems represent just one construct ofmany that can be utilized. There are a wide range of catabolic plasmidsavailable such as NAH, OCT and TOL, and other genes such as ligninperoxidases that degrade specific classes of hazardous organiccompounds, and many additional genes that are useful as suicidecassettes. These can be combined with the subject system in a matrix ofways to build a wide range of metabolic capabilities into theseorganisms to handle a wide range of environmental contaminationproblems.

As in most genetic containment systems, it is preferable to includeanother second gene in the system and often in the nucleic acid cassettealong with the suicide gene. The gene product for this second geneexercises control over the suicide gene, typically at thetranscriptional level, but sometimes at the translational level. In thismanner, there is increased control over expression of the suicide geneand a smaller chance of generating mutations which would render thesystem non-functional. Examples of gene products which can tightlycontrol expression are well-known to those of ordinary skill in the art.To form a genetic containment system, it is preferred that this secondgene be made to respond to specific physiological conditions of thecellular environment to stimulate cell killing. Consequently, theexpression product of this gene may be subject to a transcriptional ortranslational effector such as an activator or a repressor, a substrateanalog, an enzyme such as an RNA polymerase or another enzyme that isspecific to expression or repression of the suicide gene.

Regulatable promoters which respond to changed physiological conditionsare well-known and include inducible promoters such as a phage induciblepromoters, pH inducible promoters, nutrient inducible promoters(inducible by specific sugars or amino acids), temperature induciblepromoter (inducible with heat or cold), radiation inducible promoters(inducible with UVA, UVB, visible light or infrared light), metalinducible promoters (metallothionein inducible), hormone induciblepromoters (glucocorticoid inducible), steroid inducible promoters (e.g.the MMTV promoter), and hybrids and combinations of these promoters.Other useful promoters include promoters which respond to theconcentration of, or simply the presence or absence of, one or moreextrinsic agents in the system. Useful extrinsic and intrinsic agentsinclude substrates or products of amino acid synthesizing enzymes,nucleic acid and nucleotide synthesizing enzymes and most components ofbacterial catabolic and anabolic pathway operons. Promoters of thisgroup of genes respond to the presence or absence of amino acids, aminoacid analogs, saccharides (e.g. lactose and its analog IPTG),polysaccharides, nucleic acids and nucleotides, and nucleic acid baseconstituents (e.g. purine and pyrimidine salvage pathway substrates andenzymes). Additional extrinsic or intrinsic agents which are known ineither prokaryotes or eukaryotes include hormones, transcriptionalactivators and repressors, cytokines, toxins, petroleum-based compounds,metal containing compounds, salts, ions, enzyme substrate analogs andderivatives and combinations of these agents.

Optionally, to exercise additional control over the suicide gene, agenetic containment system of the invention may also contain a genewhich expresses an inhibitor of the transcriptional effector. Inhibitorsare preferably expressed at very low levels in the recombinant cell suchthat any leakiness of the suicide promoter does not destroy the cell,but is immediately scavenged and inhibited by the constant presence ofinhibitor substances. For example, T7 lysozyme is an excellent inhibitorof most T7 RNA polymerases and thereby prevent any T7 RNA polymeraseactivity. Other inhibitor substances include substrate analogs,competing enzymes and the like. Low levels of expression of thissubstance is not harmful to most cells and will prevent suicidal eventsfrom occurring prematurely. Preferably, the inhibitor is present or isintroduced into recombinant cells and expressed constituitively.

One preferred example of a streptavidin-based biological containmentsystem is comprised of a streptavidin gene whose expression iscontrolled by a first transcription promoter linked to the streptavidingene. The streptavidin promoter is regulated by a heterologous productwhich is expressed from another gene which has a differenttranscriptional promoter linked to the gene. This second transcriptionalpromoter is inducible by an extrinsic or intrinsic agent added to orremoved from the culture. The streptavidin gene may encode the entiremature protein of streptavidin or it may comprise only a core sequence.The amino acid sequence may be entirely homologous to the wild typestreptavidin or comprise point, deletion or other mutations convenientfor the process such as, for example, mutations that alter the affinityof biotin binding.

The regulatable promoter which is attached to the streptavidin gene maybe any transcriptional promoter whose activity can be controlled by aheterologous product. Heterologous products are expression productswhich are not normally present within the cell containing the system.Examples of various types of regulatable promoters include bacteriophagepromoters. Most of the T7 RNA polymerase enzyme promoters will only berecognized by T7 RNA polymerase. As most cells do not possess T7 RNApolymerase, only by introduction of the gene which encodes thispolymerase can the streptavidin promoter be recognized and thestreptavidin protein expressed.

Another embodiment of the invention is directed to a nucleic acid thatcontains a suicide cassette which encodes a biotin-binding protein. Thebiotin-binding protein is preferably streptavidin and is utilized foreither a suicide function or as a detection marker. The cassette may bea single nucleic acid fragment or the components of the cassette may bedistributed on multiple fragments. Preferably, the nucleic acid containconvenient restriction endonuclease sites to simplify transfer of thecassette between vectors, origins of replication for maintenance,transcriptional promoters as described above to control gene expressionand recognition sites for various enzymes to maximize expression whenstimulated such as during transcription or translation. Useful vectorsfor carrying or transferring the cassette include viral vectors foreukaryotic cells (AdV, AAV, HSV), plasmids and phage vectors forbacterial cells, and shuttle vectors such as cosmids for both.Optionally, the nucleic acid cassette may also encode inhibitors of thetranscriptional effector which can be utilized to reduce the chances ofundesired low levels of suicide gene expression. These inhibitors may beexpressed at high or low levels, depending on their effect of the hostcell and the ability of the suicide gene product to quickly andefficiently overcome the inhibition upon expression. Preferably, theinhibitor is constituitively expressed at fairly low levels.

Another embodiment of the invention is directed to recombinant cellsthat contains a biotin-binding genetic containment system. Thebiotin-binding component may be the suicide gene product or a marker forthe detection of recombinant cells. Useful cells may be prokaryotic oreukaryotic cells that require biotin or a derivative of biotin forviability, in the case of a suicide system, or any cell capable ofexpressing the biotin-binding component. Examples of useful eukaryoticcells include plant cells, insect cells, algae and mammalian cells.Examples of useful prokaryotic cells include gram-negative such as E.coli, and gram-positive cells such as B. subtilis, both of which areoften utilized as GEMs. Recombinant cells may be unicellular such aswith bacteria and yeast or multicellular such as with higher organismsprovided that the recombinant cells of the multicellular organism can beidentified. Optionally, cells may also express an inhibitor of theheterologous product as an additional control to prevent even minimaltranscription occurring from the streptavidin gene. Preferably, theinhibitor is constituitively expressed at low levels by the cell so asnot to interfere with induction of expression of the heterologousproduct.

Another embodiment of the invention is directed to couplingbiotin-binding genetic cassettes to detection systems. This allows forthe sensitive tracking of cells that, through natural or artificialmeans, express biotin, avidin or streptavidin. Environmental monitoringand detection of organisms based on the detection of streptavidin bybiosensor-based systems is a major advantage such as, for example, torapidly monitor cells using biotinylated lipid films as biosensors. Incombination with biotinylated fluorescent probes, as little as tenmicroorganisms can be detected in a sample.

Another embodiment of the invention is directed to a method forselectively killing a recombinant microorganism. Utilizing the geneticcontainment systems described, a recombinant cell containing such asystem can be selectively destroyed by activation of the suicide gene.Activation may be accomplished by the addition of extrinsic agents orhigher or lower concentrations of intrinsic agents to the cellularenvironment. Upon recognition of the specific agent or agents, the cellself destructs according to its genetic programming.

Another embodiment of the invention is directed to a method fordetecting a recombinant microorganism. A sample suspected of containinga cell containing a streptavidin-based genetic containment system isprovided. The streptavidin-based containment system is comprised of astreptavidin gene placed under the control of a regulatable promoterregulated by a heterologous product expressed from an inducible promoterwhich can be induced by an extrinsic agent or a change in concentrationof an intrinsic agent. The promoter is induced to express theheterologous product and activates transcription of the streptavidingene. The activated sample is contacted with a solid support to which isattached biotin. Cells which contain streptavidin become bound to thesolid support and can be easily detected.

A streptavidin-based containment system can be used to control thesurvival of engineered microorganisms genetically constructed to degradehazardous chemicals such as hydrocarbons, aromatic compounds andhalogenated compounds including toluates, xylenes, benzenes, polycyclichydrocarbons and derivatives and combinations of these compounds.According to the invention, microorganisms can be genetically altered tocouple the control of degradation of different types of hazardousorganic chemicals to the survival of the organism. This control ismanifested in the self-destruction of the microorganism once the targetchemicals in the environment have been degraded. This type of geneticcontrol will function in laboratory or controlled fermentation settings,or in the field in actual bioremediation applications. The control ofsurvival is based on expression of streptavidin protein which is onlyproduced once the target chemical is degraded. Once there is no furthertarget chemical present, or a critical lower threshold is reached, thegene encoding the streptavidin protein is expressed, and the organism iskilled due to complexation of all the essential biotin vitamin in thecell with the newly expressed streptavidin. Aside from controlledfermentations and field remediation needs, this same containment orcontrol system may be useful for the delivery of live vaccines andbiopesticides, or a range of bioengineered products both in the field orin controlled laboratory environments.

Streptavidin-based containment systems of the invention are suitable forthe treatment many types of contamination including toxic spills, sludgeand activated sludge. The costs are expected to be less than haft of thecurrent technologies. Additional benefits for this approach are thenoninvasive methods of treatment, the fact that no new potentialcontaminants will be added or left in the soil or water, and the processgoes to completion for the target chemicals of interest. In addition,the proposed method is relatively simple and requires less engineeringthan conventional methods. As additional constructs are made, the scopeof target chemicals that can be treated in this manner will increase.

Streptavidin-based genetic systems are also useful for environmentalremediation. Both biological and non-biological treatments forcontaminated soils and waters can be performed and at a lower effectivecost. This same technology can also be used in biopesticide delivery orvaccine delivery as a way to control the organisms used in some of theseprocesses. In addition, many suicide functions are being developed tocontrol genetic elements such as vital and retroviral vectors, duringgene therapy for medical treatments. The system described herein isadaptable for these applications as well. This technology can also beused to control cell lines involved in the production of importantpharmaceuticals or drugs. It is often critical to control organisms infermentation systems to assure that all organisms are killed after theiruseful life cycle in the production process. Should viable cells getout, carefully selected strains could be lost. Thus, the geneticcontainment system described may also be useful as a security system toprovide enhanced assurance that there is no chance of release of viableorganisms once growth is completed on a particular substrate.

The following experiments are offered to illustrate embodiments of theinvention, and should not be viewed as limiting the scope of theinvention.

EXAMPLES Example 1 Bacterial Strains, Plasmids and Culture Conditions.

Subcloning experiments were performed in E. coli DH5α (recA1 hsdR17endA1 thi-1 gyrA96 relA1 supE44.o slashed.80δlacZΔM15γ⁻) (T. Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1982). Suicide constructs were tested in thementioned E. coli strain and Pseudomonas putida KT2440 (hsdR1 hsdM⁺)(F.C.H. Franklin et al., Proc. Natl. Acad. Sci. USA 18:7458-62, 1981).DNA used were plasmids pKK223-3 (Amp^(r)) (J. Brosius et at., Proc.Natl. Acad. Sci. USA 6929-33, 1984), pLysE (Cm_(r)) (F. W. Studier etat., Methods Enzymol. 185:60-89, 1990), pRO1614 (Amp^(r), Tet_(r)) (R.H. Olsen et at., J. Bacteriol. 150:60-69, 1982), pTSA-13 (Amp^(r)),pUC19 (Amp^(r)) (C. Yanisch-Peron et at., Gene 33:103-19, 1985), pVLT33(Kan^(r)) (V. de Lorenzo et at., Gene 123:17-24, 1993), replicative form(rf) of bacteriophage mGP1-2, a derivative of pGP1-2 (S. Tabor et al.,Proc. Natl. Acad. Sci. USA 82:1074-78, 1985), and syntheticoligonucleotides.

Bacteria were routinely grown at 30° C. in LB or M9/glucose mediumsupplemented with 1 mM thiamine for E. coil. Modified M9/glucose mediumfor P. putida was supplemented with A9 micronutrients (M.-A. Abril etal., J. Bacteriol. 171:6782-90, 1989). Antibiotics were used at thefollowing concentrations: ampicillin and carbenicillin, 100 μg/ml (E.coil); kanamycin, 25 μg/ml (E. coil) or 75 μg/ml (P. putida);tetracycline, 10 μg/ml (E. coli). Isopropyl β-D-thiogalactopyranoside(IPTG) was used at a concentration of 1 mM.

DNA manipulations were carried out by standard procedures (T. Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1982). P. putida was transformed by a RbClmethod (M. Bagdasarian et al., Current Topics in Microbiology andImmunology, Springer-Verlag, P. M. Hofschneider and W. Goebel editors,1982) or by electroporation (efficiency˜10⁷ transformants/μg DNA on GenePulser apparatus with Pulse Controller, Bio-Rad, set at 25 μF, 2.50 kV,and 200 Ω for a 0.2 cm cuvette).

Example 2 Design of a Lethal Cassette.

To employ streptavidin as a suicide component in biological containmentsystem, the stv gene can be fused with the regulated promoter P_(lac) orits derivatives, P_(UV5), P_(tac), etc. Transcription of the stv genecould be repressed by E. coli LacI protein synthesized uponenvironmental induction. A potential drawback of such a design is aninsufficient repression of most of P_(lac) type promoters by LacIprotein. Together with high toxicity of streptavidin, this will causefailure of the whole system due to accumulation of mutants, or poorkilling efficiency in case of huge overproduction of LacI repressor.

To achieve tighter control of streptavidin synthesis, the stv gene wasplaced under the regulation of the bacteriophage T7 expression system(T. Sano et al., Proc. Natl. Acad. Sci. USA 87:142-46, 1990). In thiscase, transcription of the stv gene is controlled by the T7.o slashed.10promoter which is recognized only by T7 RNA polymerase. Bacteriophagepolymerase is provided from the T7 gene 1, placed under control of theP_(tac) promoter, negatively regulated by the LacI repressor. Tocompensate for the leakiness of P_(tac), an inhibitor of T7 RNApolymerase, T7 lysozyme (B. A. Moffatt et al., Cell 49:221-27, 1987),was also supplied. It was expressed from the T7 gene 3.5 placed underthe P. putida TOL plasmid P_(m), promoter. In an uninduced state in E.coli, P_(m) behaves as a very weak constitutive promoter, allowingsufficient synthesis of T7 lysozyme to inactivate low levels of T7 RNApolymerase. The P_(m) promoter was chosen because leaving the lysozymegene even without promoter appeared to be sufficient to reduce leakinessof P_(tac). Higher levels of lysozyme in the system are designed torespond to low levels P_(m) promoter activator which completelyrepresses basal T7 RNA polymerase expression. Streptavidin is expressedupon inactivation of LacI with IPTG, or, if the lacI gene is fused to,for example, the P_(m) promoter, in response to depletion of ahydrocarbon effector.

With this design, lethal expression of the stv gene is tightlycontrolled by the bacteriophage T7 transcription system, that is the .oslashed.10 promoter, the RNA polymerase encoded by the T7 gene 1 fusedwith Escherichia coli P_(tac) promoter, and the lysozyme, here as aninhibitor of RNA polymerase. This entire containment system can beconditioned by the E. coli lacI repressor gene fused with a promoterresponding to environmental or physiological changes. A plasmid-basedconstruct was examined in E. coli and Pseudomonas putida. Induction ofstv gene expression resulted in cell-killing with efficiency up to99.9%. Mutants escaping killing appeared at frequencies reaching 10⁻⁶-10⁻⁷ per cell per generation. The general requirement for biotin in theliving systems makes the stv cassette a candidate for containmentstrategies in a broad range of microorganisms.

Example 3 Construction of a Streptavidin System.

P_(tac) :: T7 gene I fusion: The T7 RNA polymerase gene was derived frommGP1-2 rf DNA by cleavage with EcoR I and Pst I, and subcloned into theEcoR I and Pst I sites of pKK223-3.

P_(m) :: T7 gene 3.5 fusion: A P. putida TOL meta-cleavage pathway P_(m)promoter region (B. Kessler et at., J. Mol. Biol. 230:699-703, 1993) wasassembled from two complementary 110-base oligonucleotides designed tohave EcoR I and Sac I ends after annealing. It was inserted into theEcoR I and Sac I sites of pUC19. The T7 lysozyme gene was cut out frompLysE with BamH I, blunt-ended with Klenow polymerase, and inserted intothe Sma I site of the pUC19 derivative, downstream from P_(m).

T7 gene 3.5 - stv gene cluster: The pUC19 derivative bearing the T7lysozyme gene was cut with BamH I, blunt-ended with Klenow polymerase,and then cut with EcoR I. A fragment containing the lysozyme gene fusedthe P_(m) promoter was inserted into the EcoR I and EcoR V sites ofpTSA-13, a pET-3a derivative with the stv gene encoding the corestreptavidin (amino acids 16-133 of the mature protein) under thecontrol of the T7.o slashed.10 promoter.

stv gene - T7 gene 3.5 - T7 gene I cassette: A fragment of a pTSA-13derivative containing the T7 lysozyme and stv genes with theirregulatory sequences was cut out with EcoR I and Bgl II, and blunt-endedwith Klenow polymerase. This blunt-ended fragment was inserted into theblunt-ended BamH I site of the pKK derivative described above, upstreamfrom the P_(tac) :: T7 gene I fusion. A Sph I-Hind III fragment of theresulting plasmid pKK-slp bearing the stv cassette (FIG. 1) was finallyinserted into the Sph I and Hind III sites of pRO1614, a vector forenteric bacteria and pseudomonads.

Example 4 Functionality Tests of stv Gene-based Constructs.

Functionality of the stv gene-based design was analyzed in E. coli byconstructing a DH50α strain carrying plasmids pKK-slp or pRO-sip. P.putida, which does not contain the lacI gene on the chromosome, wasfirst transformed with pVLT33 (lacA) and then with pRO-sip. However, inP. putida such construct did not respond to IPTG, probably because ofoverproduction of LacI. Similar behavior of P. putida has been alreadyreported. To be able to check the stv cassette in P. putida, it wasinserted into a multiple cloning site of a pVLT33 derivative with lacIgene depleted from its own promoter. In this case (pVLT-lslp)transcription of lacI was very low, originating somewhere within the stvcassette. Cultures were grown overnight in LB medium supplemented withappropriate antibiotic, diluted with the same medium to A₄₅₀ nm ˜0.1,and further grown to early exponential phase. As shown in FIGS. 2 and 3,the addition of IPTG at A₄₅₀ ˜0.4 inhibited cell growth, both in E. coliand P. putida.

Example 5 Efficiency of Streptavidin Killing Function.

Luria-Delbruck experiments (S. E. Luria et at., Genetics 28:491-511,1943) were performed using 96-well microtiter plates and carbenicillininstead of ampicillin (L. B. Jensen et at., Appl. Environ. Microbiol.59:3713-17, 1993). The efficiency of killing of host cells by theexpression of streptavidin was tested by counting viable cells beforeaddition of IPTG, and at different times after. Bacterial samples werewashed with LB medium to remove IPTG, spread on LB agar platessupplemented with appropriate antibiotic and 50 μg/ml biotin, andincubated for a week. An hour after induction of streptavidin synthesis,99.9% of E. coli and 92.3% of P. putida cells could not recover evenafter prolonged incubation in the presence of biotin (FIG. 3). Survivingcolonies of E. coli contained a fully active suicide system, as checkedon plates with and without IPTG. A progressive growth up of bacterialpopulation after about 10 hours of exposure to IPTG resulted from theloss of the plasmid due to destruction of ampicillin by β-lactamasereleased to the medium. In case of P. putida, all surviving clonesappeared to contain mutations (probably point mutations) within thesuicide system. That is why in this case cultures resumed just 3 hoursafter induction of the stv gene. Rates of mutational inactivation of thestreptavidin-based suicide system in E. coli DH5α and P. putida KT2440are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Fluctuation Test Results                                                      Strain           Mutation Rates of stv Constructs                             ______________________________________                                        E. coli DH5α (pRO-slp)                                                                   3.2 × 10.sup.-6                                        E coli DH5α (pVLT33, pRO-slp)                                                            2.1 × 10.sup.-7                                        P. putdia KT2440 (pVLT01slp)                                                                   2.0 × 10.sup.-4                                        ______________________________________                                    

Mutation rates in E. coli were comparable to those published for otherconstructs with a single toxic gene. The higher rate observed for P.putida is apparently due to an insufficient repression of the P_(tac)promoter in the uninduced state on account of a very low level of LacIsynthesis.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. The specification and examples shouldbe considered exemplary only with the true scope and spirit of theinvention indicated by the following claims.

We claim:
 1. A genetic comment system comprising a cassette that encodesa biotin-binding component.
 2. The containment system of claim 1 whereinthe biotin-binding component is a protein selected from the groupconsisting of streptavidin, avidin and mutants thereof.
 3. Thecontainment system of claim 2 wherein the streptavidin mutation is acore streptavidin protein.
 4. The genetic containment system of claim 1wherein the cassette comprises a regulatable promoter and a suicidegene.
 5. The containment system of claim 4 wherein the regulatablepromoter is functionally linked to said suicide gene.
 6. The containmentsystem of claim 4 wherein the suicide gene is selected from the groupconsisting of E. coli gef genes, Serratia marcescens and Staphylococcusaureus endonucleases, Serratia liquefaciens phospholipase A, Bacillussubtilis sacB gene, lysis genes from bacteriophages and combinationsthereof.
 7. The containment system of claim 4 wherein the suicide geneencodes said biotin-binding component.
 8. The containment system ofclaim 4 wherein the regulatable promoter is selected from the groupconsisting of bacteriophage SP6 promoters, bacteriophage T7 promoters,bacteriophage T3 promoters, bacteriophage γ p_(L) promoters, trppromoters, lac promoters, hybrid trp-lac promoters, phoA promoters, galpromoters, metallothionein promoters, MMTV promoters and hybrids andcombinations thereof.
 9. The containment system of claim 4 wherein theregulatable promoter is an inducible promoter selected from the groupconsisting of phage inducible promoters, nutrient inducible promoters,temperature inducible promoter, radiation inducible promoters, metalinducible promoters, hormone inducible promoters, steroid induciblepromoters and hybrids and combinations thereof.
 10. The containmentsystem of claim 4 wherein the regulatable promoter is regulated by atranscriptional effector.
 11. The containment system of claim 10 whereinthe transcriptional effector is a transcriptional repressor or atranscriptional activator.
 12. The containment system of claim 10wherein the transcriptional effector is an RNA polymerase.
 13. Thecontainment system of claim 10 wherein the regulatable promoter is abacteriophage T7 promoter and the transcriptional effector is a T7 RNApolymerase.
 14. The containment system of claim 10 wherein thetranscriptional effector is encoded within said genetic containmentsystem.
 15. The containment system of claim 10 wherein the regulatablepromoter is functionally linked to a genetic element that is regulatedby said transcriptional effector.
 16. The containment system of claim 10wherein the regulatable promoter is induced by one or more physiologicalconditions.
 17. The containment system of claim 16 wherein thephysiological conditions are selected from the group consisting ofchanges in pH, temperature, radiation, osmotic pressure, salinegradients, cell surface binding and the concentration of one or moreextrinsic or intrinsic agents.
 18. The containment system of claim 17wherein the extrinsic agent is selected from the group consisting ofamino acids and amino acid analogs, saccharides and polysaccharides,nucleic acids, transcriptional activators and repressors, cytokines,toxins, petroleum-based compounds, metal containing compounds, salts,ions, enzyme substrate analogs and combinations thereof.
 19. Thecontainment system of claim 10 further comprising an inhibitor of thetranscriptional effector.
 20. The containment system of claim 19 whereinthe inhibitor is encoded within said genetic containment system.
 21. Thecontainment system of claim 19 wherein the inhibitor is constituitivelyexpressed.
 22. The containment system of claim 19 wherein the inhibitoris a substrate analog.
 23. The containment system of claim 19 whereinthe transcriptional effector is a T7 RNA polymerase and the inhibitor isa T7 lysozyme.
 24. A genetic containment system comprised of a cassettethat encodes a suicide gene and a marker gene that encodes abiotin-binding protein.
 25. The containment system of claim 24 whereinthe marker gene encodes streptavidin protein, avidin protein or mutantsthereof.
 26. A vector that contains a genetic containment systemcomprised of a suicide gene that encodes a biotin-binding protein whoseexpression is regulated by a transcriptional effector encoded withinsaid vector wherein expression of said transcriptional effector isregulated by a physiological condition.
 27. The nucleic acid of claim 26wherein the genetic vector is a plasmid, viral vector, cosmid, phagevector or combination thereof.
 28. The nucleic acid of claim 26 whereinthe streptavidin gene is functionally linked to a regulatable promoter.29. The nucleic acid of claim 28 wherein the regulatable promoter isselected from the group consisting of bacteriophage SP6 promoters,bacteriophage T7 promoters, bacteriophage T3 promoters, bacteriophage γp_(L) promoters, trp promoters, lac promoters, hybrid trp-lac promoters,phoA promoters, gal promoters, metallothionein promoters, MMTV promotersand combinations thereof.
 30. The nucleic acid of claim 28 wherein theregulatable promoter is an inducible promoter selected from the groupconsisting of phage inducible promoters, nutrient inducible promoters,temperature inducible promoter, metal inducible promoters, hormoneinducible promoters, steroid inducible promoters and combinationsthereof.
 31. The nucleic acid of claim 26 wherein the physiologicalcondition is selected from the group consisting of a changes in pH,temperature, radiation, osmotic pressure, saline gradients andconcentration of an extrinsic or intrinsic agent.
 32. The nucleic acidof claim 31 wherein the extrinsic or intrinsic agent is selected fromthe group consisting of amino acids and amino acid analogs, saccharidesand polysaccharides, nucleic acids, transcriptional activators andrepressors, cytokines, toxins, petroleum-based compounds, metalcontaining compounds, salts, ions, enzyme substrate analogs andcombinations thereof.
 33. The nucleic acid of claim 31 wherein theextrinsic agent is IPTG or an analog thereof.
 34. The nucleic acid ofclaim 26 further comprising an inhibitor of the transcriptionaleffector.
 35. The nucleic acid of claim 34 wherein the inhibitor isconstituitively expressed.
 36. The nucleic acid of claim 34 wherein thetranscriptional effector is T7 RNA polymerase and the inhibitor is T7lysozyme.
 37. A recombinant cell containing a genetic containment systemcomprised of a suicide cassette that encodes a biotin-binding protein.38. The cell of claim 37 which is a prokaryotic or a eukaryotic cell.39. The cell of claim 38 wherein the eukaryotic cell is a plant cell, analgae cell or a mammalian cell.
 40. The cell of claim 38 wherein theprokaryotic cell is a gram-negative or a gram-positive bacterial cell.41. A genetic containment system comprising a suicide gene encoding abiotin-binding protein functionally linked to an inducible promoter, asuicide control gene encoding a heterologous polymerase functionallylinked to a repressible promoter regulated by a repressor, and apolymerase control gene functionally linked to a constitutive promoter.42. The genetic containment system of claim 41 wherein the suicide geneencodes streptavidin, avidin or mutants.
 43. The genetic containmentsystem of claim 41 wherein the inducible promoter is a bacteriophage γ,SP6, T3 or T7 promoter.
 44. The genetic containment system of claim 41wherein the heterologous polymerase is a bacteriophage, SP6, T3 or T7polymerase.
 45. The genetic containment system of claim 41 wherein therepressor is a lactose, galactose or tryptophan repressor protein. 46.The genetic containment system of claim 41 wherein the repressiblepromoter is P_(tac), P_(lac), P_(trp), P_(gal), P_(phoA) or a hybridthereof.
 47. The genetic containment system of claim 41 wherein thepolymerase control gene encodes a lysozyme.
 48. The genetic containmentsystem of claim 41 wherein the constitutive promoter is P_(m).
 49. Thegenetic containment system of claim 41 further comprising a geneencoding the repressor functionally linked to another induciblepromoter.
 50. The genetic containment system of claim 49 wherein theanother inducible promoter that is activated or repressed in response toa change of an environmental condition.
 51. The genetic containmentsystem of claim 50 wherein the environmental condition is the change inconcentration of a chemical, metal, radiation or nutrient or change inpH.
 52. A genetic containment system comprising a suicide gene encodinga biotin-binding protein functionally linked to an inducible promoter, asuicide control gene encoding a polymerase functionally linked to arepressible promoter, a polymerase control gene functionally linked to aconstitutive promoter, and a gene that encodes said repressorfunctionally linked to another inducible promoter that responds to anenvironmental condition.